Project Abstract Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), and several other cancers. Despite intensive studies, the mechanism underlying KSHV-induced cellular transformation remains unclear. This critical gap of knowledge has impeded the development of effective therapeutic approaches. KSHV genome encodes over a dozen of proteins that regulate cell proliferation, growth and survival; however, their roles in KSHV-induced oncogenesis have not been defined because of the lack of an efficient model of cellular transformation of primary cells. In the current funding period, we have made significant progresses toward this goal. We have discovered that KSHV can efficiently infect, immortalize and transform primary rat and human mesenchymal precursor cells (MM). This breakthrough, for the first time, has made it possible to delineate viral genes and cellular pathways required for KSHV-induced cellular transformation and tumorigenesis. Using this novel model combining with a reverse genetic approach, we have found that, in contrast to results of previous single gene expression studies, in the context KSHV infection viral vCyclin (ORF72) is required for cell cycle progression and cell proliferation in contact-inhibited condition but not in normal cell proliferative condition. Significantly, vCyclin promotes KSHV-induced cellular transformation and tumorigenesis. Furthermore, we have recently developed a unique Crispr-Cas9 library, performed a preliminary screening, and identified numerous cellular genes and pathways that are essential for KSHV-induced cellular transformation. Based on these preliminary studies, we have formulated a working hypothesis that KSHV induces cellular transformation and tumorigenesis by encoding specific viral genes to manipulate essential cellular gene(s)/pathway(s). We will carry out two integrated and synergistic Specific Aims: (1) To identify viral genes required for KSHV-induced cellular transformation and tumorigenesis, and define their mechanisms of action; and (2) To delineate the mechanism by which vCyclin mediates KSHV-induced cellular transformation and tumorigenesis. Upon completion of this project, our expectations are to identify the viral essential genes required for KSHV-induced cellular transformation, and define the mechanisms of action with focus on vCyclin. Together, these results will be highly significant and innovative because we will use a novel KSHV transformation model of primary cells and a novel Crispr-Cas9 screening approach to delineate viral oncogenes and cellular genes/pathways essential for cellular transformation. These studies will provide insights into the oncogenesis of KSHV-induced cancers and possibly other cancers, and identify novel targets for developing innovative therapeutic approaches for KSHV-induced malignancies.